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Correspondence: F.A. Fonseca, Diviso de Cardiologia, Departamento de Medicina, UNIFESP, Rua Pedro de Toledo, 276,
04039-030 So Paulo, SP, Brasil. Fax: +55-11-5084-8777. E-mail: fahfonseca@terra.com.br or ffonseca@cardiol.br
Received February 17, 2012. Accepted May 10, 2012. Available online July 20, 2012.
Brazilian Journal of Medical and Biological Research Online Provisional Version
ISSN 0100-879X
This Provisional PDF corresponds to the article as it appeared upon acceptance.
Fully formatted PDF and full text (HTML) versions will be made available soon.
Differences in synthesis and absorption of
cholesterol of two effective lipid-lowering therapies
S.H. Kasmas
1
, M.C. Izar
1,2
, C.N. Frana
1
, S.C. Ramos
1
, F.T. Moreira
1
, T. Helfenstein
1,2
,
R.A. Moreno
3
, N.C. Borges
3
, A.M. Figueiredo-Neto
2
and F.A. Fonseca
1,2
1
Diviso de Cardiologia, Departamento de Medicina, Universidade Federal de So Paulo, So Paulo, SP, Brasil
2
Instituto Nacional de Cincia e Tecnologia de Fluidos Complexos, So Paulo, SP, Brasil
3
Synchrophar, Campinas, SP, Brasil
Abstract
Effective statin therapy is associated with a marked reduction of cardiovascular events. However, the explanation for full ben-
efts obtained for LDL cholesterol targets by combined lipid-lowering therapy is controversial. Our study compared the effects
of two equally effective lipid-lowering strategies on markers of cholesterol synthesis and absorption. A prospective, open label,
randomized, parallel design study, with blinded endpoints, included 116 subjects. We compared the effects of a 12-week treat-
ment with 40 mg rosuvastatin or the combination of 40 mg simvastatin/10 mg ezetimibe on markers of cholesterol absorption
(campesterol and -sitosterol), synthesis (desmosterol), and their ratios to cholesterol. Both therapies similarly decreased total
and LDL cholesterol, triglycerides and apolipoprotein B, and increased apolipoprotein A1 (P < 0.05 vs baseline for all). Simvas-
tatin/ezetimibe increased plasma desmosterol (P = 0.012 vs baseline), and decreased campesterol and -sitosterol (P < 0.0001
vs baseline for both), with higher desmosterol (P = 0.007) and lower campesterol and -sitosterol compared to rosuvastatin,
(P < 0.0001, for both). In addition, rosuvastatin increased the ratios of these markers to cholesterol (P < 0.002 vs baseline for
all), whereas simvastatin/ezetimibe signifcantly decreased the campesterol/cholesterol ratio (P = 0.008 vs baseline) and tripled
the desmosterol/cholesterol ratio (P < 0.0001 vs baseline). The campesterol/cholesterol and -sitosterol/cholesterol ratios
were lower, whereas the desmosterol/cholesterol ratio was higher in patients receiving simvastatin/ezetimibe (P < 0.0001 vs
rosuvastatin, for all). Pronounced differences in markers of cholesterol absorption and synthesis were observed between two
equally effective lipid-lowering strategies.
Key words: Campesterol; -sitosterol; Desmosterol; Statin; Ezetimibe
Introduction
A recent meta-analysis has shown that marked reductions in LDL cholesterol with statins are related to a greater
absolute decrease in major cardiovascular events (1). The most appropriate strategies for achieving greater effective-
ness in cholesterol reduction include the use of high doses of potent statins or the combination of lipid-lowering drugs
(2,3). However, depending on the choice of drugs, signifcant differences in cholesterol absorption and synthesis can
be expected (4,5).
Indeed, reduction in the endogenous synthesis of cholesterol by statins can be accompanied by increased intestinal
sterol absorption (6). On the other hand, ezetimibe, an inhibitor of dietary and biliary intestinal cholesterol absorption,
has been extensively used as a lipid-lowering agent, but its role in endogenous cholesterol synthesis is less studied,
especially when combined with a statin (7).
Differences in the pharmacokinetic properties of statins may account for the effectiveness of cholesterol synthesis
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inhibition as well as for the stimuli of cholesterol absorption. Ezetimibe has an estimated half-life of approximately 22 h
(8), while simvastatin was reported to have a relatively short half-life of less than 3 h (9).
Recently, the phenotype of high absorption and low synthesis of cholesterol has been related to increased cardio-
vascular and all-cause mortality (10). On the other hand, some additional benefts of statins, including anti-infammatory
and antithrombotic properties, or improvement of endothelial function are related to the lower expression of intermediate
compounds of the endogenous cholesterol synthesis pathway (11-13).
Therefore, the overall beneft from lipid-lowering therapy for cardiovascular disease prevention seems to depend on
the effective reduction in both endogenous cholesterol synthesis and intestinal absorption of sterols. Since simvastatin
is a statin with a short half-life and lower affnity for hydroxymethylglutaryl-coenzyme A reductase than the latest genera-
tion of statins (14,15), we hypothesized that this statin could be insuffcient to counterbalance the stimulus of cholesterol
synthesis promoted by ezetimibe.
Thus, we compared the effects of two potent and equally effective lipid-lowering strategies on markers of cholesterol
synthesis and absorption.
Material and Methods
Design and study population
We performed a prospective, randomized, open-label study, with parallel arms and blinded endpoints. Patients were
recruited from the outpatient dyslipidemia unit of the Universidade Federal de So Paulo. The trial protocol was conducted
in accordance with the ethical standards of the the Universidade Federal de So Paulo on human experimentation and
approval was obtained from the Universidade Federal de So Paulo Ethics Committee. Written informed consent was
obtained from all subjects prior to inclusion.
Eligible patients were men and women, 30 to 75 years of age, under treatment for primary or secondary prevention of
coronary heart disease, who had an indication for lipid-lowering therapy according to the National Cholesterol Education
Program/Adult Treatment Panel III (NCEP/ATP) guidelines (16). A total of 116 subjects completed the study protocol.
Patients with liver, renal or gastrointestinal disease, malignancies, and uncontrolled metabolic disorders that might affect
the tolerability or safety of the treatments were excluded. Exclusion criteria during the study were low adherence (less
than 80%) to the lipid-lowering regimen. The major characteristics of the study population are listed in Table 1. Risk
factors were defned by the NCEP/ATP III guidelines (16). Before treatment, all patients received nutrition counseling
based on the Therapeutic Lifestyle Changes of the NCEP/ATP III (16). They were then randomized to receive 40 mg
rosuvastatin or the combination of 40 mg simvastatin plus 10 mg ezetimibe, daily for 12 weeks. During the study, patients
were evaluated at 4-week intervals, when compliance with study medication was confrmed. The patients did not take
lipid-lowering agents 4 weeks before the beginning of the study.
Study drugs
Rosuvastatin (Crestor

, IPR Pharmaceuticals, Porto Rico) and simvastatin/ezetimibe (Zetsim

, Schering-Plough
Products, Porto Rico) were supplied by AstraZeneca (Brazil) and Merck Co. (Brazil), respectively.
Blood sample collection and assays
Biochemical analyses were performed in samples obtained after a 12-h fasting period at baseline and after 12
weeks of study treatment and were processed by a central laboratory of the Universidade Federal de So Paulo using
automated techniques.
Lipids and biochemistry
Serum total cholesterol, HDL cholesterol, and triglycerides were determined by automated methods (Advia 2400, Sie-
mens Healthcare Diagnostics, Japan). LDL cholesterol was calculated using the Friedewald equation (17). Apolipoprotein
A1, apolipoprotein B, and highly sensitive C-reactive protein (hs-CRP) were determined by nephelometry (Array 360 CE/
AL, Beckmann Coulter, Inc., USA). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and
creatine kinase (CK) were assayed by automated techniques (Advia 2400, Siemens Healthcare Diagnostics).
Markers of sterol absorption and synthesis
For the quantifcation of -sitosterol and campesterol (markers of sterol absorption), as well as for desmosterol (a
precursor of endogenous cholesterol synthesis), we used ultra-performance liquid chromatography (UPLC) and mass
spectrometry (MS) as previously reported (18,19). Briefy, the method consisted of liquid-liquid extraction followed by
separation in the UPLC system and detection with an atmospheric pressure chemical ionization (APCI) ion source mass
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spectrometer operating on single ion monitoring for each sterol (-sitosterol, campesterol, and desmosterol). The MS
system (Quattro Premier-XE, Waters Co., UK) was adjusted to monitor single ions formed by an APCI ion source. The
sterols were detected as their free forms, i.e., non-esterifed, with the ions being monitored with a mass to charge ratio
(m/z) of 367.30 for desmosterol, 397.25 for -sitosterol, and 383.60 for campesterol. Absolute values of plasma sterol
levels were reported as mg/dL. As these sterols are transported by lipoproteins, absolute values of sterols were corrected
for total plasma cholesterol and reported as their ratios to cholesterol.
Statistical analyses
Numerical data are reported as means SEM or medians (interquartile range). Categorical data are reported as
number (%) and compared by the Pearson chi-square test. Continuous variables were tested for distribution of normality
by the Kolmogorov-Smirnov test. Variables with Gaussian distribution were compared between times and groups using
a general linear model (GLM) - repeated measures. When data were not normally distributed, we used Mann-Whitney
or Wilcoxon tests for comparisons between groups and within groups, respectively. Statistical signifcance was set at P
< 0.05. All analyses were two-tailed and were carried out using the SPSS 17.0 for Windows (SPSS Inc., USA).
Results
Patients
A total of 116 subjects presented >80% adherence to the study protocol. The major characteristics of these subjects
were comparable between groups at baseline (Table 1).
Lipids, apolipoproteins, and other laboratory parameters
At baseline, biochemical analyses including lipid profle and apolipoproteins did not differ between groups (Table 2).
A 12-week treatment with either 40 mg rosuvastatin or 40 mg simvastatin/10 mg ezetimibe promoted similar reductions
of total and LDL cholesterol, as well as of serum triglyceride levels, without changes in HDL cholesterol. Both regimens
increased serum apolipoprotein A1 and reduced serum apolipoprotein B levels without differences between treatment
arms (Table 2).
Creatinine and glucose did not differ between groups along the study. AST, ALT and CK were slightly increased
after the treatments, but remained within the normal range. hs-CRP levels decreased similarly in each treatment group
(Table 3).
Campesterol, -sitosterol and desmosterol
At baseline, plasma levels of campesterol, -sitosterol and desmosterol were comparable between groups. Treatment
with high-dose rosuvastatin (40 mg) did not change the plasma markers of sterol absorption and endogenous cholesterol
synthesis over a period of 12 weeks (P = NS, Wilcoxon test) (Table 4, Figure 1A-C). Conversely, in subjects receiving
40 mg simvastatin/10 mg ezetimibe, we observed increased cholesterol synthesis, as shown by the increase in plasma
desmosterol levels (P = 0.012) and by the decrease in cholesterol absorption markers (P < 0.0001 for both campesterol
and -sitosterol) at the end of treatment compared to their baseline levels (Table 4, Figure 1A-C). In addition, patients
treated with the combination of simvastatin/ezetimibe presented lower plasma campesterol and -sitosterol levels than
those receiving rosuvastatin (P < 0.0001); these patients also presented higher plasma desmosterol levels (P = 0.007)
compared to rosuvastatin-treated subjects.
The ratios between absorption markers and cholesterol (campesterol/cholesterol, -sitosterol/cholesterol) and be-
tween synthesis markers and cholesterol (desmosterol/cholesterol) were also comparable between groups at baseline
(P = NS). Since rosuvastatin reduced total and LDL cholesterol levels, the synthesis and absorption ratios for cholesterol
were all increased over time (P < 0.002 vs baseline). Treatment with simvastatin/ezetimibe decreased the campesterol/
cholesterol ratio (P = 0.008 vs baseline), but did not change the -sitosterol/cholesterol ratio (P = 0.960 vs baseline), and
increased desmosterol/cholesterol ratio (P < 0.0001 vs baseline). In addition, the campesterol/cholesterol and -sitosterol/
cholesterol ratios were lower and the desmosterol/cholesterol ratio was higher in patients receiving simvastatin/ezetimibe
than in those treated with rosuvastatin (P < 0.0001, for all; Table 4, Figure 2A-C).
Discussion
We found important differences between the two lipid- lowering regimens in the balance of markers of sterol absorp-
tion and synthesis, in spite of a similar decrease in serum LDL cholesterol levels. Combined therapy with simvastatin
plus ezetimibe reduced the markers of cholesterol absorption and promoted an increase in plasma desmosterol levels,
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suggesting that the inhibition of endogenous cholesterol synthesis by simvastatin is not suffcient to avert the increased
endogenous synthesis following the blockade of cholesterol absorption by ezetimibe. Conversely, treatment with ro-
suvastatin resulted in a similar decrease in cholesterol levels without changes in absolute campesterol, -sitosterol or
desmosterol values. However, due to the higher degree of cholesterol synthesis inhibition observed with potent statins
alone, the ratios between markers of absorption and synthesis and cholesterol differed between treatment groups, showing
exacerbation of the effect of ezetimibe increasing endogenous cholesterol synthesis and attenuating the expected inhibi-
tion of cholesterol absorption when used in combination with simvastatin. The reduction in LDL cholesterol observed with
lipid-lowering agents per se is a stimulus for the increase in cholesterol synthesis, regardless of the mechanism by which
cholesterol is reduced. Therefore, when combining a statin with a short half-life with an inhibitor of cholesterol absorption
with a long half-life, we can expect a more pronounced increase in cholesterol synthesis markers. Conversely, if a potent
statin with a long half-life is used alone, a more prominent inhibition of cholesterol synthesis can be expected.
Interestingly, the effects on intestinal absorption markers may differ among statins. The maximal doses of atorvastatin
and rosuvastatin were compared in a study in which the authors found a modest absolute decrease in campesterol and
sitosterol levels with rosuvastatin, and an increase in these cholesterol absorption markers with atorvastatin (5). The
authors postulated an interaction between ABCG5/G8 transporters and atorvastatin to explain their fndings (20). Other
factors, such as the presence of diabetes, have been reported to modify the magnitude of cholesterol synthesis inhibition
following lipid-lowering therapies (21).
The relationship between phytosterols and the severity of coronary disease was described in the Ludwigshafen Risk
and Cardiovascular Health (LURIC) study (22), which reported data for 2440 individuals in whom coronary angiograms
and sterol levels were evaluated. The phenotype of low cholesterol synthesis combined with high cholesterol absorption
showed an association with severity of coronary disease. In agreement with these fndings, the Framingham Offspring
Study also showed that the low-synthesis and high-absorption phenotypes were independent predictors of cardiovascular
disease (23). In addition, these markers of absorption and synthesis are also related to HDL cholesterol concentrations
(24).
Phytosterolemia is a rare genetic disorder characterized by a substantial increase in plasma levels of plant sterols
and premature atherosclerosis (25,26). However, the role of modest increases in plasma phytosterol levels in the risk
for coronary disease is controversial (22,27-30).
Reduction of sterol absorption can prevent the increase in cholesterol and plasma plant sterol levels. However, the
results of our study suggest the need for an effective statin to avoid the increase in endogenous cholesterol synthesis
by the inhibition of cholesterol absorption.
In our study, all patients received nutrition counseling according to NCEP III guidelines (16). A novel mechanism for
the reduction of intestinal sterol absorption following the intake of plant sterols has been recently described. Interestingly,
despite the competition between cholesterol and plant sterols for incorporation into micelles, -sitosterol was reported to
reduce the expression of the Niemann-PickC1-like 1 (NPC1L1) transporter (31). Therefore, in our study, a less pronounced
increase in phytosterol levels may have been infuenced by the adherence of these subjects to the diet in both arms.
Recently, a meta-analysis of data from 170,000 subjects in 26 randomized studies confrmed the beneft and safety of
LDL cholesterol lowering using statins, and suggested that the reduction of 2-3 mM would reduce major cardiovascular
events by about 40-50% (1). On the other hand, the benefts of LDL cholesterol lowering using other strategies seem
less clear (32-34).
Our results showed that two highly effective lipid-lowering strategies induced a similar reduction of LDL cholesterol
levels. However, substantial differences were found in the metabolism of sterols. Since differences in cholesterol ho-
meostasis characterized by the balance between sterol absorption and synthesis are related to the severity of coronary
disease, these differences may explain the unexpected fndings in recent clinical trials. Further studies addressing car-
diovascular events are needed to assess the impact of such differences on sterol metabolism.
Acknowledgments
Research supported by FAPESP (#2008/55443-6) and CNPq (#2008/57685-7). S.H. Kasmas was the recipient of a
grant from FAPESP (#2008/52597-2).
References
1. Baigent C, Blackwell L, Emberson J, Holland LE, Reith C, Bhala N, et al. Effcacy and safety of more intensive lowering of LDL
cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials. Lancet 2010; 376: 1670-1681.
2. Jones PH, Davidson MH, Stein EA, Bays HE, McKenney JM, Miller E, et al. Comparison of the effcacy and safety of rosuvastatin
www.bjournal.com.br Braz J Med Biol Res Online Provisional Version
O
N
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N
E

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R
O
V
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S
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versus atorvastatin, simvastatin, and pravastatin across doses (STELLAR* Trial). Am J Cardiol 2003; 92: 152-160.
3. Ballantyne CM, Weiss R, Moccetti T, Vogt A, Eber B, Sosef F, et al. Effcacy and safety of rosuvastatin 40 mg alone or in combi-
nation with ezetimibe in patients at high risk of cardiovascular disease (results from the EXPLORER study). Am J Cardiol 2007;
99: 673-680.
4. Assmann G, Kannenberg F, Ramey DR, Musliner TA, Gutkin SW, Veltri EP. Effects of ezetimibe, simvastatin, atorvastatin, and
ezetimibe-statin therapies on non-cholesterol sterols in patients with primary hypercholesterolemia. Curr Med Res Opin 2008;
24: 249-259.
5. van Himbergen TM, Matthan NR, Resteghini NA, Otokozawa S, Ai M, Stein EA, et al. Comparison of the effects of maximal dose
atorvastatin and rosuvastatin therapy on cholesterol synthesis and absorption markers. J Lipid Res 2009; 50: 730-739.
6. Jakulj L, Vissers MN, Groen AK, Hutten BA, Lutjohann D, Veltri EP, et al. Baseline cholesterol absorption and the response to
ezetimibe/simvastatin therapy: a post-hoc analysis of the ENHANCE trial. J Lipid Res 2010; 51: 755-762.
7. Jakulj L, Vissers MN, van Roomen CP, van der Veen, Vrins CL, Kunne C, et al. Ezetimibe stimulates faecal neutral sterol excre-
tion depending on abcg8 function in mice. FEBS Lett 2010; 584: 3625-3628.
8. Kosoglou T, Statkevich P, Johnson-Levonas AO, Paolini JF, Bergman AJ, Alton KB. Ezetimibe: a review of its metabolism, phar-
macokinetics and drug interactions. Clin Pharmacokinet 2005; 44: 467-494.
9. Lennernas H, Fager G. Pharmacodynamics and pharmacokinetics of the HMG-CoA reductase inhibitors. Similarities and differ-
ences. Clin Pharmacokinet 1997; 32: 403-425.
10. Matthan NR, Resteghini N, Robertson M, Ford I, Shepherd J, Packard C, et al. Cholesterol absorption and synthesis markers
in individuals with and without a CHD event during pravastatin therapy: insights from the PROSPER trial. J Lipid Res 2010; 51:
202-209.
11. Wang CY, Liu PY, Liao JK. Pleiotropic effects of statin therapy: molecular mechanisms and clinical results. Trends Mol Med 2008;
14: 37-44.
12. Liu PY, Liu YW, Lin LJ, Chen JH, Liao JK. Evidence for statin pleiotropy in humans: differential effects of statins and ezetimibe
on rho-associated coiled-coil containing protein kinase activity, endothelial function, and infammation. Circulation 2009; 119:
131-138.
13. Zhou Q, Liao JK. Pleiotropic effects of statins. - Basic research and clinical perspectives -. Circ J 2010; 74: 818-826.
14. Bellosta S, Paoletti R, Corsini A. Safety of statins: focus on clinical pharmacokinetics and drug interactions. Circulation 2004;
109: III50-III57.
15. Rubba P, Marotta G, Gentile M. Effcacy and safety of rosuvastatin in the management of dyslipidemia. Vasc Health Risk Manag
2009; 5: 343-352.
16. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of
High Blood Cholesterol in Adults (Adult Treatment Panel III) fnal report. Circulation 2002; 106: 3143-3421.
17. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without
use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499-502.
18. Ramos SC, Fonseca FA, Kasmas SH, Moreira FT, Helfenstein T, Borges NC, et al. The role of soluble fber intake in patients
under highly effective lipid-lowering therapy. Nutr J 2011; 10: 80.
19. Feio CA, Izar MC, Ihara SS, Kasmas SH, Martins CM, Feio MN, et al. Euterpe oleracea (Acai) modifes sterol metabolism and
attenuates experimentally-induced atherosclerosis. J Atheroscler Thromb 2011 (in press).
20. Kajinami K, Brousseau ME, Nartsupha C, Ordovas JM, Schaefer EJ. ATP binding cassette transporter G5 and G8 genotypes
and plasma lipoprotein levels before and after treatment with atorvastatin. J Lipid Res 2004; 45: 653-656.
21. Guyton JR, Betteridge DJ, Farnier M, Leiter LA, Lin J, Shah A, et al. Achievement of recommended lipid and lipoprotein levels
with combined ezetimibe/statin therapy versus statin alone in patients with and without diabetes. Diab Vasc Dis Res 2011; 8:
160-172.
22. Silbernagel G, Fauler G, Renner W, Landl EM, Hoffmann MM, Winkelmann BR, et al. The relationships of cholesterol metabolism
and plasma plant sterols with the severity of coronary artery disease. J Lipid Res 2009; 50: 334-341.
23. Matthan NR, Pencina M, LaRocque JM, Jacques PF, DAgostino RB, Schaefer EJ, et al. Alterations in cholesterol absorption/
synthesis markers characterize Framingham offspring study participants with CHD. J Lipid Res 2009; 50: 1927-1935.
24. Nunes VS, Leanca CC, Panzoldo NB, Parra E, Cazita PM, Nakandakare ER, et al. HDL-C concentration is related to markers of
absorption and of cholesterol synthesis: Study in subjects with low vs. high HDL-C. Clin Chim Acta 2011; 412: 176-180.
25. Bhattacharyya AK, Connor WE. Beta-sitosterolemia and xanthomatosis. A newly described lipid storage disease in two sisters.
J Clin Invest 1974; 53: 1033-1043.
26. Salen G, Horak I, Rothkopf M, Cohen JL, Speck J, Tint GS, et al. Lethal atherosclerosis associated with abnormal plasma and
tissue sterol composition in sitosterolemia with xanthomatosis. J Lipid Res 1985; 26: 1126-1133.
27. Assmann G, Cullen P, Erbey J, Ramey DR, Kannenberg F, Schulte H. Plasma sitosterol elevations are associated with an in-
creased incidence of coronary events in men: results of a nested case-control analysis of the Prospective Cardiovascular Munster
(PROCAM) study. Nutr Metab Cardiovasc Dis 2006; 16: 13-21.
28. Miettinen TA, Gylling H. The effects of statins and sitosterols: beneft or not? Curr Atheroscler Rep 2009; 11: 23-27.
29. Strandberg TE, Gylling H, Tilvis RS, Miettinen TA. Serum plant and other noncholesterol sterols, cholesterol metabolism and
22-year mortality among middle-aged men. Atherosclerosis 2010; 210: 282-287.
30. Izar MC, Tegani DM, Kasmas SH, Fonseca FA. Phytosterols and phytosterolemia: gene-diet interactions. Genes Nutr 2011; 6:
17-26.
31. Jesch ED, Seo JM, Carr TP, Lee JY. Sitosterol reduces messenger RNA and protein expression levels of Niemann-Pick C1-like
www.bjournal.com.br Braz J Med Biol Res Online Provisional Version
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P
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1 in FHs 74 Int cells. Nutr Res 2009; 29: 859-866.
32. Rossebo AB, Pedersen TR, Boman K, Brudi P, Chambers JB, Egstrup K, et al. Intensive lipid lowering with simvastatin and
ezetimibe in aortic stenosis. N Engl J Med 2008; 359: 1343-1356.
33. Kastelein JJ, Akdim F, Stroes ES, Zwinderman AH, Bots ML, Stalenhoef AF, et al. Simvastatin with or without ezetimibe in familial
hypercholesterolemia. N Engl J Med 2008; 358: 1431-1443.
34. Taylor AJ, Villines TC, Stanek EJ, Devine PJ, Griffen L, Miller M, et al. Extended-release niacin or ezetimibe and carotid intima-
media thickness. N Engl J Med 2009; 361: 2113-2122.
Table 1. Baseline characteristics of the study population according to
treatment assignment.
Characteristic Rosuvastatin
(N = 58)
Simvastatin/ezetimibe
(N = 58)
Age, years, median (IQ) 59 (53-65) 59 (54-65)
Male gender, N (%) 17 (29.3) 18 (31.0)
Diabetes mellitus, N (%) 12 (20.7) 14 (24.1)
Hypertension, N (%) 46 (79.3) 44 (75.9)
Smoking, N (%) 5 (8.6) 4 (6.9)
Metabolic syndrome, N (%) 35 (60.3) 27(46.6)
Primary prevention, N (%) 47 (81.0) 48 (82.8)
There were no statistical differences between groups (chi-square test).
IQ = interquartile range. Metabolic syndrome according to the National
Cholesterol Education Program/Adult Treatment Program III (NCEP/ATP
III) guidelines (16).
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Table 2. Lipid and apolipoprotein parameters of the two groups at
baseline and at the end of the study.
Parameter Rosuvastatin
(N = 58)
Simvastatin/ezetimibe
(N = 58)
P (wg)
Cholesterol
Baseline 250 6 243 6 <0.0001
End of study 144 4 150 6
LDL-C
Baseline 161 5 160 5 <0.0001
End of study 67 3 76 4
HDL-C
Baseline 54 2 52 2 0.38
End of study 53 2 52 2
Triglycerides
Baseline 173 11 152 9 <0.0001
End of study 117 6 110 6
Apo A1
Baseline 150 4 142 3 0.02
End of study 152 4 148 3
Apo B
Baseline 131 4 133 4 <0.0001
End of study 67 2 71 3
Lipids and apolipoproteins are reported as means SEM in mg/dL.
LDL-C and HDL-C = low- and high-density lipoprotein cholesterol,
respectively; Apo = apolipoprotein. There were signifcant statistical
differences within groups (wg) (general linear model with repeated
measures test); however, there were no signifcant differences be-
tween groups (general linear model with repeated measures test).
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Table 3. Laboratory parameters by groups at baseline and at the end of
the study.
Parameter Rosuvastatin
(N = 58)
Simvastatin/ezetimibe
(N = 58)
P (wg)
Creatinine (mg/dL)
Baseline 0.93 0.03 0.92 0.03 0.99
End of study 0.91 0.03 0.94 0.03
AST (IU/L)
Baseline 22 (19-25) 22 (19-25) 0.001
R
End of study 25 (20-32) 24 (21-29)
ALT (IU/L)
Baseline 20 (16-25) 21 (17-30) 0.001
R
End of study 23 (17-37) 27 (19-32) <0.0001
S/E
CK (IU/L)
Baseline 104 (84-159) 127 (81-167) <0.0001
R
End of study 135 (106-204) 118 (95-193) 0.02
S/E
hs-CRP (mg/L)
Baseline 2.7 (1.2-5.4) 2.7 (1.6-5.4) <0.0001
R
End of study 1.5 (0.9-3.5) 1.6 (0.7-3.2) <0.0001
S/E
Glucose
Baseline 97 (88-110) 97 (86-107) 0.67
R
End of study 97 (90-105) 97 (89-110) 0.09
S/E
AST = aspartate aminotransferase; ALT = alanine aminotransferase; CK =
creatine kinase; hs-CRP = highly sensitive C-reactive protein. Creatinine is
reported as means SEM and was analyzed by the general linear model
with repeated measures. AST, ALT, CK, hs-CRP, and glucose are report-
ed as median and interquartile range and were not normally distributed.
They were analyzed by the Wilcoxon test (for within-group comparisons)
or the Mann-Whitney test (for between-group comparisons). There were
signifcant statistical differences within groups (wg); however, there were
no signifcant differences between groups.
R
Comparisons for rosuvastatin
between visits;
S/E
comparisons for simvastatin/ezetimibe between visits.
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Table 4. Markers of cholesterol absorption and synthesis and their ratios to cholesterol by groups at
baseline and at the end of the study.
Parameter Rosuvastatin Simvastatin/ezetimibe P (bg) P (wg)
Campesterol (mg/dL)
Baseline
Absolute 0.97 (0.57-1.48) 0.89 (0.53-1.51) 0.732 0.263
R
Ratio to cholesterol 0.0037 (0.0024-0.0060) 0.0035 (0.0024-0.0070) 0.808 <0.0001
R
End of study
Absolute 0.96 (0.76-1.48) 0.40 (0.29-0.57) <0.0001 <0.0001
S/E
Ratio to cholesterol 0.0072 (0.0053-0.0093) 0.0028 (0.0019-0.0040) <0.0001 0.008
S/E
-sitosterol (mg/dL)
Baseline
Absolute 0.66 (0.42-1.00) 0.61 (0.40-1.06) 0.910 0.382
R
Ratio to cholesterol 0.0027 (0.0018-0.0039) 0.0029 (0.0016-0.0043) 0.791 <0.0001
R
End of study
Absolute 0.72 (0.51-0.99) 0.40 (0.26-0.58) <0.0001 <0.0001
S/E
Ratio to cholesterol 0.0050 (0.0036-0.0066) 0.0030 (0.0018-0.0040) <0.0001 0.960
S/E
Desmosterol (mg/dL)
Baseline
Absolute 0.39 (0.15-0.85) 0.33 (0.14-1.02) 0.987 0.972
R
Ratio to cholesterol 0.0014 (0.0006-0.0031) 0.00 14 (0.0005-0.0034) 0.886 0.001
R
End of study
Absolute 0.43 (0.22-0.72) 0.62 (0.45-1.02) 0.007 0.012
S/E
Ratio to cholesterol 0.0033 (0.0017-0.0051) 0.0046 (0.0027-0.0071) 0.010 <0.0001
S/E
Data as reported as medians (25th-75th percentiles).
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Comparisons for rosuvastatin between visits;
S/
E
comparisons for simvastatin/ezetimibe between visits. There were signifcant statistical differences both
between (bg; Mann-Whitney test) and within groups (wg; Wilcoxon test).
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Figure 1. Box-plots (median, 25th and 75th percentiles) for plasma levels of campesterol (A), -sitosterol (B), and desmosterol (C) at
baseline and at the end of the study according to treatment. Baseline values were comparable between groups. Rosuvastatin did not
modify these parameters over time. Subjects treated with simvastatin/ezetimibe presented a reduction of plasma levels of campesterol
and -sitosterol (P < 0.0001 vs baseline for both, Wilcoxon test), and an increase of desmosterol (P = 0.012 vs baseline, Wilcoxon test).
Plasma campesterol and -sitosterol levels were lower at the end of the study (P < 0.0001 for both, Mann-Whitney test) in subjects
receiving simvastatin/ezetimibe compared to individuals receiving rosuvastatin, whereas desmosterol levels were higher (P = 0.007,
Mann-Whitney test).
Figure 2. Box-plots (median, 25th and 75th percentiles) of the campesterol/cholesterol (A), -sitosterol/cholesterol (B) and desmos-
terol/cholesterol (C) ratios at baseline and at the end of the study according to treatment. Baseline values were comparable between
groups. Rosuvastatin increased all of these ratios (P < 0.002 vs baseline, for all, Wilcoxon test). Treatment with simvastatin/ezetimibe
promoted a signifcant decrease of the campesterol/cholesterol ratio (P = 0.008 vs baseline, Wilcoxon test), but not of the -sitosterol/
cholesterol ratio (P = NS). Conversely, the desmosterol/cholesterol ratio increased 3-fold after treatment with simvastatin/ezetimibe (P
< 0.0001). Comparison of the effects of treatments showed that campesterol/cholesterol and -sitosterol/cholesterol ratios were lower
in patients receiving simvastatin/ezetimibe than in those treated with rosuvastatin at the end of treatment (P < 0.0001, Mann-Whitney
test), whereas the desmosterol/cholesterol ratio was higher in patients treated with simvastatin/ezetimibe (P < 0.0001).